Codon optimized il-15 and il-15r-alpha genes for expression in mammalian cells

ABSTRACT

The present invention provides for nucleic acids improved for the expression of interleukin-15 (IL-15) in mammalian cells. The invention further provides for methods of expressing IL-15 in mammalian cells by transfecting the cell with a nucleic acid sequence encoding an improved IL-15 sequence. 
     The present invention further provides expression vectors, and IL-15 and IL-15 receptor alpha combinations (nucleic acid and protein) that increase IL-15 stability and potency in vitro and in vivo. The present methods are useful for the increased bioavailability and biological effects of IL-15 after DNA, RNA or protein administration in a subject (e.g. a mammal, a human).

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is the continuation of U.S. patent applicationSer. No. 12/160,263, filed Jul. 8, 2008, which is U.S. National Stageentry of International Application No. PCT/US07/00774, filed Jan. 12,2007, which claims the benefit of U.S. Provisional Patent ApplicationNos. 60/812,566, filed on Jun. 9, 2006 and 60/758,819, filed on Jan. 13,2006, the entire contents of each of which are hereby incorporatedherein by reference for all purposes

REFERENCE TO SEQUENCE LISTING SUBMITTED AS TEXT FILE

This application includes a Sequence Listing as a text file named“77867-946361-SEQLIST.txt” created Jun. 11, 2015, and containing 80,299bytes. The material contained in the text file is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to improved cytokine expression inmammalian cells by optimizing all steps of gene expression of thecytokine.

BACKGROUND OF THE INVENTION

Interleukin-15 (IL-15) is a pleiotropic cytokine important for both theinnate and adaptive immune systems (Diab, et al., Cytotherapy (2005)7:23-35). IL-15 promotes the activation of neutrophils and macrophages,and is essential to the development and function of dendritic cells(DC), natural killer (NK) cells, NK T cells, and CD8+ T cells (Id.).IL-15 acts on cells in both lymphoid and non-lymphoid compartments (VanBelle and Grooten, Arch Immunol Ther Exp (2005) 53:115).

Based on its many functions and relative safety in animal models,administration of IL-15 finds use in treating immunodeficiency, for thein vitro expansion of T cells and NK cells, and as an adjuvant forvaccines, including anti-HIV vaccines (Diab, et al., supra; Ahmad, etal., Curr HIV Res (2005) 3:261; Alpdogan and van den Brink, TrendsImmunol (2005) 26:56). For example, administration of exogenous IL-15has been found to drastically enhance the immune cell functions of humanimmunodeficiency virus (HIV)-infected Acquired Immune DeficiencySyndrome (AIDS) patients (Ahmad, et al., supra; see also, Pett andKelleher, Expert Rev Anti Infect Ther (2003) 1:83; and Ansari, et al.,Immunol Res (2004) 29:1). Administration of IL-15 for its effects onlymphopoiesis and the treatment of immunodeficiency disorders is alsobeing explored (Alpdogan and van den Brink, supra).

Results from several investigators have suggested that the naturalsoluble form of IL-15 Receptor alpha is an antagonist of IL-15 (see,Mortier, et al., (2004) J. Immunol. 173, 1681-1688; Ruchatz, et al.,(1998) J. Immunol. 160, 5654-566; and Smith, et al., (2000) J. Immunol.165, 3444-3450). In contrast, the sushi domain of IL-15 Receptor alphawhen fused to IL-15 via a flexible amino acid linker has been proposedas an agonist of IL-15 function in vitro (J Biol Chem. 2006 Jan. 20;281(3):1612-9). Soluble interleukin-15 receptor alpha (IL-15Ralpha)-sushi is a selective and potent agonist of IL-15 action throughIL-15R beta/gamma (see, Mortier E, et al., J Biol Chem. 2006 281:1612).

To provide therapeutic IL-15, alone or in combination with a whole IL-15receptor alpha or a soluble IL-15 receptor alpha, either foradministration as a coding nucleic acid or as a protein, it is importantto develop efficient expression vectors and efficiently expressioncoding nucleic acid sequences for this cytokine. The present inventionaddresses this need.

BRIEF SUMMARY OF THE INVENTION

The present invention provides nucleic acid sequences, expressionvectors and mammalian cells for the high-level expression ofinterleukin-15 (IL-15), alone and combined with whole IL-15 Receptoralpha (IL15Ra) or the soluble form of IL15Ra (IL15sRa). The inventionfurther provides methods for the high-level expression of interleukin-15in mammalian cells, alone and combined with whole IL-15 Receptor alpha(IL15Ra) or the soluble form of IL15Ra (IL15sRa).

In a related aspect, the invention provides nucleic acid sequences,expression vectors and mammalian cells for the high-level expression ofwhole IL-15 Receptor alpha (IL15Ra) or the soluble form of IL15Ra(IL15sRa). The invention further provides methods for the high-levelexpression whole IL-15 Receptor alpha (IL15Ra) or the soluble form ofIL15Ra (IL15sRa).

In one aspect, the invention provides nucleic acid sequences encoding aninterleukin-15 (IL-15) protein having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity to a native mammalian IL-15protein, wherein the nucleic acid sequence differs from a nucleic acidsequence encoding the native mammalian IL-15 by at least 50% of thechanged nucleotide positions identified in FIG. 8. In some embodiments,the nucleic acid sequence differs from a nucleic acid sequence encodingthe native mammalian IL-15 by at least 50% of the changed codonpositions identified in FIG. 4 and/or in FIG. 6. In some embodiments,the changed nucleotides and codons are in the mature IL-15 sequence. Thenative mammalian IL-15 can be any mammalian IL-15, including humanIL-15, a primate IL-15, a porcine IL-15, a murine IL-15, and the like.

In some embodiments, the nucleic acid sequence encoding the IL-15differs from a nucleic acid sequence encoding the native IL-15 by atleast about 55% (e.g., 59 nucleotides), 60% (e.g., 64 nucleotides), 65%(e.g., 70 nucleotides), 70% e.g., (75 nucleotides), 75% (e.g., 81nucleotides), 80% (e.g., 86 nucleotides), 85% (e.g., 91 nucleotides),90% (e.g., 97 nucleotides), 95% (e.g., 109 nucleotides) of the 115changed nucleotide positions identified in FIG. 8 (shaded). In someembodiments, the nucleic acid sequence encoding the IL-15 differs from anucleic acid sequence encoding the native IL-15 by at least about 55%(e.g., 66 codons), 60% (e.g., 73 codons), 65% (e.g., 78 codons), 70%e.g., (85 codons), 75% (e.g., 91 codons), 80% (e.g., 97 codons), 85%(e.g., 103 codons), 90% (e.g., 109 codons), 95% (e.g., 115 codons) ofthe 121 changed codon positions identified in FIG. 4 (shaded, boxed orunderlined).

In some embodiments, the changed nucleotides and codons are in themature IL-15 sequence. For example, the nucleic acid sequence encodingthe improved IL-15 can differ from a nucleic acid sequence encoding thenative IL-15 by at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% of the78 changed nucleotide positions in the mature IL-15 identified in FIG. 8(shaded). In another embodiment, the nucleic acid sequence encoding theimproved IL-15 can differ from a nucleic acid sequence encoding thenative IL-15 by at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% of the84 changed codon positions in the mature IL-15 identified in FIG. 4(shaded, boxed or underlined).

In some embodiments, the nucleic acid sequence differs from a nucleicacid sequence encoding the native IL-15 at nucleotide positions 6, 9,15, 18, 21, 22, 27, 30, 33, 49, 54, 55, 57, 60, 63, 69, 72, 75, 78, 81,84, 87, 90, 93, 96, 105, 106, 114, 120, 123, 129, 132, 135, 138, 141,156, 159, 162, 165, 168, 169, 174, 177, 180, 183, 186, 189, 192, 195,198, 204, 207, 210, 213, 216, 217, 219, 222, 228, 231, 237, 246, 252,255, 258, 261, 277, 283, 285, 291, 294, 297, 300, 306, 309, 312, 315,318, 321, 324, 327, 330, 333, 336, 339, 351, 354, 363, 364, 369, 372,375, 384, 387, 390, 393, 396, 402, 405, 414, 423, 426, 429, 432, 435,438, 442, 450, 453, 456, 459, 462, 468, 483 and 486, wherein thenucleotide positions are as identified in FIG. 8.

In some embodiments, the nucleic acid sequence comprises a guanine (g)or a cytosine (c) nucleotide at nucleotide positions 6, 9, 15, 18, 21,22, 27, 30, 33, 49, 54, 55, 57, 60, 63, 69, 72, 75, 78, 81, 84, 87, 96,105, 106, 114, 120, 123, 129, 132, 135, 138, 141, 156, 159, 162, 165,168, 169, 174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210,213, 216, 217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277,283, 285, 291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327,330, 333, 336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390,393, 396, 402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453,456, 459, 462, 468, 483 and 486, wherein the nucleotide positions are asidentified in FIG. 8.

The codons can differ in any way such that an identical or similar(i.e., conservatively substituted) amino acid is encoded. In someembodiments, the codons are changed to increase GC content. In someembodiments, the improved IL-15 nucleic acid sequences each comprise atleast about 50%, 55%, 60%, 65%, 70%, 75% or more GC content (e.g.,deoxyguanosine and deoxycytidine deoxyribonucleoside residues orguanosine and cytidine ribonucleoside residues) over the length of thesequence.

The nucleic acid encoding the IL-15 can share at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a nucleic acidof SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:16. In some embodiments,the nucleic acid sequence encoding the IL-15 differs from a nucleic acidsequence encoding the native IL-15 as identified in FIG. 8 (SEQ ID NO:3or SEQ ID NO:4) or FIG. 16 (SEQ ID NO:16).

In some embodiments, the nucleic acid sequence encoding an IL-15 signalpeptide-propeptide (SIG-PRO) is replaced with a nucleic acid sequenceencoding a signal peptide (SIG) or a signal peptide-propeptide (SIG-PRO)from a heterologous protein. In some embodiments, the nucleic acidsequence encoding an IL-15 signal peptide is replaced with a nucleicacid sequence encoding a signal peptide from a heterologous protein. Theheterologous protein can be, for example, from tissue plasminogenactivator (tPA), growth hormone, granulocyte-macrophage colonystimulating factor (GM-CSF) or an immunoglobulin (e.g., IgE). In oneembodiment, the nucleic acid sequence encoding an IL-15 signalpeptide-propeptide (SIG-PRO) is replaced with a nucleic acid sequenceencoding a tPA SIG-PRO having 95% sequence identity to SEQ ID NO:6, SEQID NO:8, SEQ ID NO:25 or SEQ ID NO:27. In some embodiments, the nucleicacid encoding the IL-15 is operably linked to a nucleic acid encoding anRNA export element, for example a CTE or RTEm26CTE.

In some embodiments, the nucleic acid sequence encoding an IL15Ra signalpeptide is replaced with a nucleic acid sequence encoding a signalpeptide (SIG) or a signal peptide-propeptide (SIG-PRO) from aheterologous protein. In some embodiments, the nucleic acid sequenceencoding an IL15Ra signal peptide is replaced with a nucleic acidsequence encoding a signal peptide from a heterologous protein. Theheterologous protein can be, for example, from tissue plasminogenactivator (tPA), growth hormone, granulocyte-macrophage colonystimulating factor (GM-CSF) or an immunoglobulin (e.g., IgE). In someembodiments, the nucleic acid encoding the IL15Ra is operably linked toa nucleic acid encoding an RNA export element, for example a CTE orRTEm26CTE.

In another aspect, the invention provides nucleic acid sequencesencoding a signal peptide-propeptide (SIG-PRO) sequence from a proteinother than IL-15, for example a tPA SIG-PRO sequence, a growth hormonesignal sequence (SIG), an immunoglobulin signal sequence (SIG), operablylinked to a nucleic acid encoding an IL-15 protein having at least 90%sequence identity to the native mammalian IL-15 protein, wherein thenucleic acid sequence encoding IL-15 comprises at least 50% GC content.In one embodiment, the SIG-PRO sequence is from a protein selected fromthe group consisting of tPA, GM-CSF, growth hormone and animmunoglobulin family protein. In one embodiment, the SIG-PRO sequenceencodes a tPA SIG-PRO having at least 95% amino acid sequence identityto SEQ ID NO:6 or SEQ ID NO:8. In another embodiment, the SIG-PROsequence is a tPA SIG-PRO having at least 95% nucleic acid sequenceidentity to SEQ ID NO:5 or SEQ ID NO:7. Further embodiments are asdescribed above.

In a further aspect, the invention includes expression vectors andmammalian cells comprising the nucleic acid sequences of the invention,including the embodiments described above.

In some embodiments, the nucleic acid sequences encoding the IL-15and/or IL15Ra further include pharmaceutical excipients for use as avaccine adjuvant. In some embodiments, the nucleic acid sequencesencoding the IL-15 and/or IL15Ra further include pharmaceuticalexcipients for use as an immunotherapy factor, for example, in theexpansion of the numbers of lymphocytes, including B-cells, T cells, NKcells, and NK T cells, in vitro or in vivo. In some embodiments, theIL-15and/or IL15Ra nucleic acid sequences are used to expand lymphocytepopulations that express the IL-2/IL-15 beta gamma receptors. In someembodiments, the IL-15and/or IL15Ra nucleic acid sequences are used toexpand CD4+ and/or CD8+ T cells. In some embodiments, the IL-15and/orIL15Ra nucleic acid sequences are used to expand the numbers of dualsecreting IL-2 and IFN-gamma multifunctional cells (e.g.,multifunctional T cells) after antigenic stimulation.

In a another aspect, the invention provides methods of expressing IL-15in a mammalian cell, the method comprising recombinantly modifying amammalian cell to express a nucleic acid encoding an IL-15 proteinhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to a native mammalian IL-15 protein, wherein thenucleic acid sequence differs from a nucleic acid sequence encoding thenative mammalian IL 15 by at least 50% of the nucleotide positionsidentified in FIG. 8. The embodiments for the methods are as describedabove for the nucleic acid sequences.

In a related aspect, the present invention is based, in part, on thediscovery that the whole IL-15 Receptor alpha (IL15Ra) or the solubleform of IL15Ra (IL15sRa) comprising the entire extracellular domain ofthe receptor is a potent stabilizer of IL-15 in vitro and in vivo. Thecomplex of IL-15 and IL15sRa has increased stability in circulation andalso has increased IL-15 potency as determined by the expansion ofmultiple lymphocyte subsets including natural killer (NK) cells and Tcells. The present invention provides methods, expression vectors andprotein combinations that increase IL-15 potency in vitro and in vivo.These methods are useful for the increased bioavailability, stability,and potency of IL-15, and for increasing the biological effects of IL-15upon administration to an individual (e.g., a mammal, a human).

Provided are expression vectors for the co-ordinate expression of IL-15with its receptor IL-15 Receptor alpha (IL15Ra). The vectors generallycontain one copy of an IL-15 coding sequence or/and one copy of an IL-15Receptor alpha (IL15Ra) (whole or soluble). The expression ratios of thetwo proteins can be adjusted to 1:1, 1:2 or 1:3, for example, by usingdifferent plasmid DNA ratios (w/w) or by selecting promoters ofdifferent expression strengths. In some embodiments, the IL-15 cytokineand IL-15 Receptor alpha (IL15Ra) are expressed in a molar ratio of 1:3.

In one embodiment, the nucleic acid sequences for at least one of theIL-15 cytokine and IL-15 Receptor alpha (IL15Ra) are improved inaccordance with the present methods described herein. Co-expression ofthe IL-15 cytokine and IL-15 Receptor alpha (IL15Ra), whole or soluble,increases the amount of IL-15 cytokine and IL15Ra that is expressed andsecreted from a cell, by more than 10-fold, 100-fold, 10,000-fold,100,000-fold, 1,000,000-fold or more, in comparison to expression ofIL-15 alone, particularly in comparison to wt IL-15 sequences. Usingsuch vectors increases the stability of IL-15 and IL15Ra by more than10-fold, 20-fold, 50-fold, 100-fold, 1000-fold or more, in comparison toIL-15 alone, and increases the steady-state levels of IL-15 protein invivo. The biological function (e.g., the activation and induction of theexpansion of lymphocytes, including B cells, T cells, natural killer(NK) cells and NK T cells) of IL-15 co-expressed with IL15Ra is alsodramatically increased in vivo, by more than 10-fold, 15-fold, 20-fold,or more, in comparison to IL-15 alone. These vectors are useful for theincreased delivery of biologically active cytokines in specific tissues.The IL-15 and IL15Ra vectors and proteins find use in prophylactic andtherapeutic vaccinations, cancer immunotherapy, or for any indicationfor enhanced lymphocyte numbers and function and any immune deficiencyconditions.

In one aspect, the present invention provides expression vectors for thecoordinate expression of IL-15 with whole IL15Ra or soluble IL15Ra. TheIL-15 and whole IL15Ra or soluble IL15Ra can be contained in the sameexpression vector or in multiple expression vectors. In someembodiments, the coding nucleic acid sequence of at least one of theIL-15 and whole IL15Ra or soluble IL15Ra is improved according to thepresent methods for high efficiency expression.

One aspect of the invention is that the provided vectors expressingIL-15 and full length IL15Ra upon delivery to a mammalian cell or amammal can rapidly generate the native form of soluble extracellularIL15sRa. Therefore, co-delivery and expression of IL-15 and IL15Ragenerates IL-15/IL-15R complexes on the surface of the cell as well asIL-15/IL15sRa complexes that are released into circulation and can actat distant tissues.

In a further aspect, the invention provides improved nucleic acidsequences encoding a whole IL-15 Receptor alpha (IL15Ra) or the solubleform of IL15Ra (IL15sRa) having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity to a native mammalian IL-15Receptor alpha (IL15Ra) or the soluble form of IL15Ra (IL15sRa) protein(see, e.g., NM_002189), wherein the nucleic acid sequence differs from anucleic acid sequence encoding the native mammalian IL-15 by at least50% of the changed nucleotide positions identified in FIGS. 35-38.

In some embodiments, the coding sequence for the IL15Ra (whole orsoluble form) shares at least 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity with a nucleic acid sequence depicted in any one of FIGS.35-38. In one embodiment, the IL15Ra is encoded by the nucleic acidsequence depicted in any one of FIGS. 35-38. In one embodiment, theimproved IL15Ra (whole or soluble) coding nucleic acid sequence has atleast 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% GC content.

The invention further provides methods of increasing IL-15 quantity,stability and bioactivity. The methods can be carried out in vitro byco-expressing IL-15 and IL15Ra or IL15sRa in mammalian host cells. Themethods can be carried out in vivo by administering to an individual acombination of IL-15 with an IL-15 receptor alpha (whole or soluble), asproteins for injection or as DNA constructs (native or improved) thatare produced in vivo. One or both of the IL-15 and IL15Ra codingsequences can be improved according to the methods described herein.

The invention further provides host cells and cell lines thatcoordinately produce IL-15 and IL-15 soluble Receptor alpha (IL15sRa) orcell lines coordinately producing IL-15 and a mixture of soluble andfull length IL15Ra.

In a further aspect, the invention provides methods of enhancing theimmune response of an individual against one or more antigens byadministering an improved IL-15 nucleic acid of the invention, alone orin combination with an IL15Ra. The IL15Ra can be in protein or nucleicacid form, wild-type or improved.

In a further aspect, the invention provides methods of expanding thenumbers of lymphocytes, for example, for decreasing immunodeficiencyconditions, in vivo or in vitro, by administering an improved IL-15nucleic acid of the invention, alone or in combination with an IL15Ra.The IL15Ra can be in protein or nucleic acid form, wild-type orimproved. In some embodiments, the lymphocytes are selected from thegroup consisting of B-cells, T cells, NK cells, and NK T cells. In someembodiments, the IL-15and/or IL15Ra nucleic acid sequences promote theexpansion of lymphocyte populations that express the IL-2/IL-15 betagamma receptors. In some embodiments, the IL-15and/or IL15Ra nucleicacid sequences stimulate the expansion of CD4+ and/or CD8+ T cells. Insome embodiments, the IL-15and/or IL15Ra nucleic acid sequences inducethe expansion of the numbers of dual secreting IL-2 and IFN-gammamultifunctional cells (e.g., multifunctional T cells) upon antigenstimulation.

In some embodiments, one or both of the DNA constructs are administeredby injection and/or electroporation. Administration by dual routes ofinjection and electroporation can be done concurrently or sequentially,at the same or different sites.

DEFINITIONS

The term “native mammalian interleukin-15 (IL-15)” refers to anynaturally occurring interleukin-15 nucleic acid and amino acid sequencesof the IL-15 from a mammalian species. Those of skill in the art willappreciate that interleukin-15 nucleic acid and amino acid sequences arepublicly available in gene databases, for example, GenBank through theNational Center for Biotechnological Information on the worldwideweb atncbi.nlm.nih.gov. Exemplified native mammalian IL-15 nucleic acid oramino acid sequences can be from, for example, human, primate, canine,feline, porcine, equine, bovine, ovine, rodentia, murine, rat, hamster,guinea pig, etc. Accession numbers for exemplified native mammalianIL-15 nucleic acid sequences include NM_172174 (human; SEQ ID NO:1);NM_172175 (human); NM_000585 (human); U19843 (macaque; SEQ ID NO:14);DQ021912 (macaque); AB000555 (macaque); NM_214390 (porcine); DQ152967(ovine); NM_174090 (bovine); NM_008357 (murine); NM_013129 (rattus);DQ083522 (water buffalo); XM_844053 (canine); DQ157452 (lagomorpha); andNM_001009207 (feline). Accession numbers for exemplified nativemammalian IL-15 amino acid sequences include NP_751914 (human; SEQ IDNO:2); CAG46804 (human); CAG46777 (human); AAB60398 (macaque; SEQ IDNO:15); AAY45895 (macaque); NP_999555 (porcine); NP_776515 (bovine);AAY83832 (water buffalo); ABB02300 (ovine); XP_849146 (canine);NP_001009207 (feline); NP_037261 (rattus); and NP_032383 (murine).

The term “interleukin-15” or “IL-15” refers to a polypeptide that has atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to a native mammalian IL-15 amino acid sequence, or anucleotide encoding such a polypeptide, is biologically active, meaningthe mutated protein (“mutein”) has functionality similar (75% orgreater) to that of a native IL-15 protein in at least one functionalassay. Exemplified functional assays of an IL-15 polypeptide includeproliferation of T-cells (see, for example, Montes, et al., Clin ExpImmunol (2005) 142:292), and activation of NK cells, macrophages andneutrophils. Methods for isolation of particular immune cellsubpopulations and detection of proliferation (i.e., ³H-thymidineincorporation) are well known in the art. Cell-mediated cellularcytotoxicity assays can be used to measure NK cell, macrophage andneutrophil activation. Cell-mediated cellular cytotoxicity assays,including release of isotopes (⁵¹Cr), dyes (e.g., tetrazolium, neutralred) or enzymes, are also well known in the art, with commerciallyavailable kits (Oxford Biomedical Research, Oxford, M; Cambrex,Walkersville, Md.; Invitrogen, Carlsbad, Calif.). IL-15 has also beenshown to inhibit Fas mediated apoptosis (see, Demirci and Li, Cell MolImmunol (2004) 1:123). Apoptosis assays, including for example, TUNELassays and annexin V assays, are well known in the art with commerciallyavailable kits (R&D Systems, Minneapolis, Minn.). See also, Coligan, etal., Current Methods in Immunology, 1991-2006, John Wiley & Sons.

The term “native mammalian interleukin-15 Receptor alpha (IL15Ra)”refers to any naturally occurring interleukin-15 receptor alpha nucleicacid and amino acid sequences of the IL-15 receptor alpha from amammalian species. Those of skill in the art will appreciate thatinterleukin-15 receptor alpha nucleic acid and amino acid sequences arepublicly available in gene databases, for example, GenBank through theNational Center for Biotechnological Information on the worldwideweb atncbi.nlm.nih.gov. Exemplified native mammalian IL-15 receptor alphanucleic acid or amino acid sequences can be from, for example, human,primate, canine, feline, porcine, equine, bovine, ovine, rodentia,murine, rat, hamster, guinea pig, etc. Accession numbers for exemplifiednative mammalian IL-15 nucleic acid sequences include NM_002189 (Homosapiens interleukin 15 receptor, alpha (IL15RA), transcript variant 1,mRNA).

The term “interleukin-15 receptor alpha” or “IL15Ra” refers to apolypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to a native mammalian IL15Ra amino acidsequence, or a nucleotide encoding such a polypeptide, is biologicallyactive, meaning the mutated protein (“mutein”) has functionality similar(75% or greater) to that of a native IL15Ra protein in at least onefunctional assay. One functional assay is specific binding to a nativeIL-15 protein.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. The term encompasses nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding properties as the reference nucleic acid, andwhich are metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Degenerate codon substitutions can beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

Degenerate codon substitutions for naturally occurring amino acids arein Table 1.

TABLE 1 1^(st) position 2^(nd) position 3^(rd) position (5′ end) U(T) CA G (3′ end) U(T) Phe Ser Tyr Cys U(T) Phe Ser Tyr Cys C Leu Ser STOPSTOP A Leu Ser STOP Trp G C Leu Pro His Arg U(T) Leu Pro His Arg C LeuPro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser U(T) Ile Thr Asn Ser CIle Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U(T) Val Ala AspGly C Val Ala Glu Gly A Val Ala Glu Gly G

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region(e.g., any one of SEQ ID NOs:1-23), when compared and aligned formaximum correspondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI web site or the like). Such sequences arethen said to be “substantially identical.” This definition also refersto, or can be applied to, the compliment of a test sequence. Thedefinition also includes sequences that have deletions and/or additions,as well as those that have substitutions. As described below, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25, 50, 75, 100,150, 200 amino acids or nucleotides in length, and oftentimes over aregion that is 225, 250, 300, 350, 400, 450, 500 amino acids ornucleotides in length or over the full-length of am amino acid ornucleic acid sequences.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared (here, an entire “nativemammalian” IL-15 amino acid or nucleic acid sequence). When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLASTalgorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST software is publicly available through theNational Center for Biotechnology Information on the worldwide web atncbi.nlm.nih.gov/. Both default parameters or other non-defaultparameters can be used. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” as used herein applies to amino acidsequences. One of skill will recognize that individual substitutions,deletions or additions to a nucleic acid, peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins        (1984)).

The term “GC content” refers to the percentage of a nucleic acidsequence comprised of deoxyguanosine (G) and/or deoxycytidine (C)deoxyribonucleosides, or guanosine (G) and/or cytidine (C)ribonucleoside residues.

The terms “mammal” or “mammalian” refer to any animal within thetaxonomic classification mammalia. A mammal can refer to a human or anon-human primate. A mammal can refer to a domestic animal, includingfor example, canine, feline, rodentia, including lagomorpha, murine,rattus, Cricetinae (hamsters), etc. A mammal can refer to anagricultural animal, including for example, bovine, ovine, porcine,equine, etc.

The term “operably linked” refers to a functional linkage between afirst nucleic acid sequence and a second nucleic acid sequence, suchthat the first and second nucleic acid sequences are transcribed into asingle nucleic acid sequence. Operably linked nucleic acid sequencesneed not be physically adjacent to each other. The term “operablylinked” also refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a transcribable nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the transcribable sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates strategies of improving the coding sequences of humaninterleukin-15 (IL-15). Section 1 of the IL-15 polypeptide refers to thesignal peptide (amino acids 1-29); section 2 refers to the propeptide(amino acids 30-47); section 3 refers to mature IL-15 (amino acids47-115). In IL-15opt1, the coding sequence for IL-15 has higher GCcontent, and potential splice sites are altered. The changes generallydo not affect coding potential. In IL-15opt2, the coding sequenceimprovements are similar to IL-15opt1, but include usage of more“preferred” codons, as defined in U.S. Pat. No. 5,786,464.

FIG. 2 illustrates a comparison of the AU-GC content profiles ofwild-type IL-15 (wt), IL-15opt1 (opt1), and IL-15opt2 (opt2). BothIL-15opt1 and IL-15opt2 have a significant increase in GC contentcompared to wild-type IL-15 cDNA.

FIG. 3 illustrates a comparison of human wild-type IL-15 (SEQ ID NO:1)and improved IL-15opt1 (opt) (SEQ ID NO:3) nucleotide sequences. Thesequences share 70.7% sequence identity.

FIG. 4 illustrates a comparison of the nucleotide changes betweenwild-type human IL-15 (top; SEQ ID NO:1) and improved human IL-15opt1(bottom; SEQ ID NO:3) nucleotide sequences. The improved human IL-15sequence was changed at 121 of 162 total codons (75%). Forty-one (41)codons were left unchanged in comparison to the wild-type human IL-15nucleotide sequence. The boxed codons indicate changes to “morepreferred” codons according to the classification of Seed (U.S. Pat. No.5,786,464) (62 codons). The underlined codons indicate codons changed to“less preferred” codons according to the classification of Seed (10codons), in contradiction to the method of Seed. The grey highlightedcodons indicate changes to “not preferred” codons (49 codons), also incontradiction to the method of Seed.

FIG. 5 illustrates a sequence alignment of the nucleic acid sequences ofwild-type IL-15 (wt) (SEQ ID NO:1), IL-15opt1 (opt) (SEQ ID NO:3), andIL-15opt2 (opt-2) (SEQ ID NO:4). Wild-type IL-15 has 162 total codons.In IL-15opt1, 121 of 162 codons are changed. In IL-15opt2, 122 of 162codons are changed.

FIG. 6 illustrates a sequence alignment of the nucleic acid sequences ofwild-type IL-15 (wt; SEQ ID NO:1), IL-15opt1 (opt; SEQ ID NO:3), andIL-15opt2 (opt-2; SEQ ID NO:4). Improvement of the coding sequencesincludes nucleotide changes that use “preferred” codons or “lesspreferred” codons, as defined in U.S. Pat. No. 5,786,464. IL-15opt1 has72 preferred/less preferred codons, and IL-15opt2 has 103 preferred/lesspreferred codons. In addition, improvements of the IL-15 codingsequences include nucleotide changes that are in contradiction to themethod defined in U.S. Pat. No. 5,786,464.

FIG. 7 illustrates that plasmids having improved human IL-15 codingsequences express increased level of human IL-15 protein in transfectedmammalian cells. Depicted is a typical experiment showing a 5-foldincrease using either the IL-15opt1 or IL-15opt2 nucleic acid sequences.Over an average of 7 experiments, a mean increase of 8-fold in humanIL-15 protein production was achieved in comparison to expression fromthe wild-type human IL-15 sequence. There is no difference in IL-15protein production between IL-15opt1 and IL-15opt2. This underscores ourconclusions that it is not codon usage but rather changes of the RNAsequence that lead to improved gene expression.

FIG. 8 illustrates the common positions of nucleotide changes(highlighted) in IL-15opt1 (SEQ ID NO:3) and IL-15opt2 (SEQ ID NO:4)sequences compared to wild type human IL-15 (SEQ ID NO:1). Changes atthe positions of the indicated 115 nucleotides (highlighted) aresufficient for improved mRNA and protein expression of human IL-15 (anapproximately 8-fold increase in comparison to wild-type human IL-15).

FIG. 9 illustrates that modification of signal and/or propeptide ofhuman IL-15 leads to an increased extracellular accumulation of IL-15.

FIG. 10 illustrates that improved IL-15 coding sequences fused to thesignal peptide and propeptide of tissue plasminogen activator (tPA)greatly improves IL-15 protein production in mammalian cells. ExpressingIL-15 from IL-15opt-tPA2, which contains tissue plasminogen activatorsignal peptide and propeptide sequences, resulted in an additional 2.5average fold increase of IL-15 protein production in mammalian cells(mean of 4 experiments using 1-3 independent clones) in comparison toimproved IL-15 (opt). Other variants with differentially swappeddomains, which either had the tPA signal peptide only (IL-15opt-tPA1) ora combination of the tPA signal peptide with the IL-15 propeptide(IL-15opt-tPA3) resulted in decreased IL-15 protein production or noimprovement.

FIG. 11 illustrates the improved expression of human IL-15-tPA2, incomparison to wild-type human IL-15 (IL-15 wt) and improved IL-15(IL-15opt), from transfected human 293 and RD4 cells.

FIG. 12 illustrates alteration of the tPA signal peptide-IL-15 junctionto produce the proper N-terminus for IL-15. IL-15opt-tPA2 sequence (SEQID NO:37) has a furin cleavage site and 4 extra amino acids (GARA; SEQID NO:41) at the N-terminus (SEQ ID NO:38) in comparison to wild-typehuman IL-15 (SEQ ID NO:5 and SEQ ID NO:6). IL-15opt-tPA5 sequence (SEQID NO:39)has a furin cleavage site sequence (R-X-(K/R)-R) and 2additional amino acids (GA) immediately adjacent to the N terminus (SEQID NO:40) of the mature IL-15 (SEQ ID NO:7 and SEQ ID NO:8). IL15opt=SEQID NO:36. The resulting IL-15 proteins were sequenced from thesupernatant of transfected 293 cells and were shown to have theindicated extra amino acids immediately adjacent to the N terminus ofmature IL-15.

FIG. 13 illustrates similar production of IL-15 protein from modifiedtPA fusion proteins, IL-15opt-tPA2 (opt-tPA2) and IL-15opt-tPA5(opt-tPAS).

FIG. 14 illustrates that human IL-15 (h-IL15) (SEQ ID NO:2) and Rhesusmonkey (Macaca mulatta) IL-15 (rh-IL15) (SEQ ID NO:15) proteins share96% identity, differing by 6 amino acids. Site-directed mutagenesis wasused to introduce the indicated 11 nucleotide changes into the humanIL-15opt1 coding nucleotide sequence, generating the Rhesus IL-15optcoding nucleotide sequence.

FIG. 15 illustrates a comparison of the nucleotide sequences of humanIL-15opt1 (huIL-15opt) (SEQ ID NO:3) and Rhesus IL-15opt (rhIL-15opt)(SEQ ID NO:16). Eleven (11) nucleotide changes were introduced into the489 nucleotide coding region of human IL-15opt1.

FIG. 16 illustrates a comparison of Rhesus wild-type IL-15 (wt) (SEQ IDNO:14) and Rhesus improved IL-15 (opt) (SEQ ID NO:16) nucleotidesequences. The nucleotide sequences share 71.3% identity.

FIG. 17 illustrates that improvement of the Rhesus IL-15 coding sequenceresulted in an approximately 30-fold increase in Rhesus IL-15 proteinproduction in mammalian cells. Substitution of the IL-15 signal peptideand propeptide sequences with tPA signal peptide and propeptidesequences resulted in a further 3-fold improvement, indicatingsynergistic effects of the two approaches.

FIG. 18 illustrates that improving IL-15 coding sequences led to greatincreases of both human and Rhesus IL-15 protein production. The finalincrease in expression was approximately 20-fold for human and a 90-100fold increase for Rhesus IL-15. In both human and Rhesus IL-15 vectors,the substitution of the IL-15 signal peptide and propeptide with tPAsignal peptide and propeptide sequences led to an additionalapproximately 3-fold increase in IL-15 protein production from mammaliancells.

FIG. 19 illustrates a schematic representation of an expression vectorfor expressing optimized human IL-15 from a cytomegalovirus promoter(CMVhuIL-15opt).

FIG. 20 illustrates a sequence map of expression vector CMVhuIL-15(opt1)(SEQ ID NO:13). See, SEQ ID NO:21 for the corresponding expressionvector for expressing optimized Rhesus IL-15 from a cytomegaloviruspromoter (CMVrhIL-15opt). Human IL-15=SEQ ID NO:2; kanamycin marker=SEQID NO:42.

FIG. 21 illustrates a schematic representation of human optimized IL-15with the signal peptide and propeptide sequences from tissue plasminogenactivator protein (huIL-15opt-tPA2).

FIG. 22 illustrates the nucleic acid sequence (SEQ ID NO:9) and aminoacid sequence (SEQ ID NO:10) of huIL-15opt1-tPA2. See, SEQ ID NO:17 andSEQ ID NO:18 for the corresponding nucleic acid and amino acid sequencesof Rhesus optimized IL-15 with the signal peptide and propeptidesequences from tissue plasminogen activator protein (rhIL-15opt-tPA2).

FIG. 23 illustrates a schematic representation of human optimized IL-15with modified signal peptide and propeptide sequences from tissueplasminogen activator protein (huIL-15opt-tPA5).

FIG. 24 illustrates the nucleic acid sequence (SEQ ID NO:11) and aminoacid sequence (SEQ ID NO:12) of huIL-15opt1-tPA5. See, SEQ ID NO:19 andSEQ ID NO:20 for the corresponding nucleic acid and amino acid sequencesof Rhesus optimized IL-15 with the signal peptide and propeptidesequences from tissue plasminogen activator protein (rhIL-15opt-tPA2).

FIG. 25 illustrates that fusion of a wild-type IL-15 sequence to an RNAexport element, including CTE or RTEm26CTE resulted in an approximately2-fold increase in IL-15 protein production from mammalian cells.

FIG. 26 illustrates that improving the human IL-15 coding sequencefurther increases IL-15 protein production from human 293 cells 5-foldas compared to wild-type human IL-15 operably linked to RNA exportelements CTE or RTEm26CTE and 10-fold as compared to wild-type humanIL-15.

FIG. 27 illustrates that improving the human IL-15 coding sequencefurther increases IL-15 protein production from 293 cells at least2-fold in comparison to wild-type human IL-15 produced from RNA exportelements CTE or RTEm26CTE.

FIG. 28 illustrates changes in the tPA-IL-15 fusion as exemplified inIL-15opt-tPA6 and IL-15opt-tPA7. IL-15opt-tPA6 contains a furin cleavagesite sequence (R-X-(K/R)-R) and the 3 additional amino acids (GAR)immediately adjacent to the N terminus of the mature IL-15 (see, SEQ IDNOs:24 and 25). IL-15opt-tPA7 contains a furin cleavage site sequence(R-X-(K/R)-R) and one additional amino acid (G) immediately adjacent tothe N terminus of the mature IL-15 (see, SEQ ID NOs:26 and 27). Theresulting IL-15 proteins were sequenced from the supernatant oftransfected 293 cells and were shown to have the indicated additionalamino acids immediately adjacent to the N terminus of mature IL-15.Peptides=SEQ ID NOS:43-46.

FIG. 29 illustrates that improved human IL-15 sequences humanIL-15opt-tPA6 and human IL-15opt-tPA7 show similar increased levels ofIL-15 production in comparison to human IL-15opt-tPA2 and humanIL-15opt-tPA5. Protein levels produced from the different improvedsequences were measured from transfected human 293 cells. The producedIL-15 proteins differ at the N terminus by either having GARA, GAR, GAor G immediately adjacent to the N terminus. Different plasmidsexpressing the tPA signal fused to N terminus of the mature IL-15 showsimilar levels of improved IL-15 production.

FIG. 30 illustrates that improved rhesus IL-15 sequences rhesusIL-15opt-tPA6 and rhesus IL-15opt-tPA7 show similar increased levels ofIL-15 production in comparison to rhesus IL-15opt-tPA2 and rhesusIL-15opt-tPA5. Protein levels produced from the different improvedsequences were measured from transfected human 293 cells. The data areanalogous to those using improved human IL-15 sequences, supra.

FIG. 31 illustrates a schematic of human IL-15opt-tPA6.

FIG. 32 illustrates the nucleic acid sequence (SEQ ID NO:28) and aminoacid sequence (SEQ ID NO:29) of human IL-15opt1-tPA6. The nucleic acidand amino acid sequences for Rhesus IL-15opt-tPA6 are shown as SEQ IDNOs:32 and 33, respectively.

FIG. 33 illustrates a schematic of human IL-15opt-tPA7.

FIG. 34 illustrates the nucleic acid sequence (SEQ ID NO:30) and aminoacid sequence (SEQ ID NO:31) of human IL-15opt1-tPA7. The nucleic acidand amino acid sequences for Rhesus IL-15opt-tPA7 are shown as SEQ IDNOs:34 and 35, respectively.

FIG. 35 illustrates the nucleic acid of an improved human IL-15 receptoralpha (IL15Ra) nucleic acid sequence (SEQ ID NO:47) and the encodedamino acid sequence (SEQ ID NO:48).

FIG. 36 illustrates the nucleic acid of an improved human IL15Ra nucleicacid sequence (SEQ ID NO:47) and the encoded amino acid sequence (SEQ IDNO:48).

FIG. 37 illustrates the nucleic acid of an improved human soluble IL15Ranucleic acid sequence (SEQ ID NO:49) and the encoded amino acid sequence(SEQ ID NO:50).

FIG. 38 illustrates the nucleic acid of an improved human soluble IL15Ranucleic acid sequence (SEQ ID NO:49) and the encoded amino acid sequence(SEQ ID NO:50).

FIG. 39 illustrates that co-expression of IL-15 with IL-15 Receptoralpha improved sequences in human 293 cells in vitro using standardtransfection methods led to a dramatic increase of total IL-15 levelsmeasured. 100 ng of hIL-15 (native (plasmid AG32) or using the tPAleader (plasmid AG59)), alone or in combination with hIL15Ra (plasmidAG79) were transfected in 293 cells together 100 ng of GFP and 100 ngSEAP by the Ca—PO₄ co-precipitation method. After 48 hours cells wereharvested and Elisa was performed using Quantikine Human IL-15, RDsystems to quantify IL-15 in media (extra) and cells. Total IL-15(extracellular and intracellular) is also indicated. Fold indicates thefold increase in IL-15.

FIG. 40 illustrates that co-expression of IL-15 with IL-15 Receptoralpha improved sequences in human 293 cells in vitro using standardtransfection methods led to a dramatic increase of total IL-15 levelsmeasured. Human 293 cells were transfected with 100 ng of plasmidhIL15-tPA6 alone or in combination with either hIL15Receptor alpha(plasmid AG79) or hIL15 soluble Receptor alpha (plasmid AG98) togetherwith 100 ng of GFP and 100 ng SEAP plasmids as transfection controlsusing Superfect. Medium was sampled after 24 and 48 hours. After 48hours cells were harvested and ELISA was performed using QuantikineHuman IL-15 (R&D systems) to measure IL-15 levels.

FIG. 41 illustrates the great increase in the levels of lung NK cellsand also increases of lung CD3+CD49+ cells when IL-15 and IL-15 receptorDNA were delivered and expressed in mice tissues after tail veininjection. Number of cells are given per 10⁵ cells in the analysis file.Tissues were analyzed 3 days after tail vein injection. The differentgroups of mice were injected in the tail vein hydrodynamically with thefollowing DNAs:

-   -   GFP, 1 μg of plasmid expressing Green Fluorescent Protein        (control);    -   IL15, 1 μg of plasmid expressing the human IL-15 using the        plasmid hIL15tPA6 described in our provisional application    -   Ra, 1 μg of plasmid expressing human IL-15 Receptor alpha    -   15+Ra 2, 2 μg of plasmid expressing IL-15tPA6 and 2 μg of        plasmid expressing human IL-15 Receptor alpha    -   15+Ra 1, 1 μg of plasmid expressing IL-15tPA6 and 1 μg of        plasmid expressing human IL-15 Receptor alpha.

FIG. 42 illustrates the increase of NK cells and T cells in the liver ofmice after DNA injection of IL-15 alone, IL-15+IL-15 Receptor alpha, orIL-15+IL-15 soluble Receptor alpha. Number of cells are given per 10⁶cells in the analysis file. Organs from mice injected with IL-15tPA6 andIL-15 Receptor alpha plasmid DNAs as indicated were digested withcollagenase to obtain single cell suspensions. The cells were stainedwith antibodies against CD3, CD4, CD8, CD49b, CD44 and CD62L andanalyzed by flow cytometry. Murine NK cells are phenotypicallyidentified as CD3-CD49b+. IL-15, injection with plasmid IL-15tPA6.IL-15+IL-15R, the plasmid expressing the full IL15Ra was co-transfected.IL-15+IL-15sR, the plasmid expressing the soluble IL-15 Receptor alphawas cotransfected with IL-15tPA6.

FIG. 43 illustrates the increase in the effector cells in the spleen(total effectors and CD8 effectors, left and right panels,respectively). The lack of CD62L defines a population of murine memory Tcells with effector phenotype. Spleens from mice injected with IL-15 andIL-15 Receptor alpha plasmid DNAs as indicated were processed and cellswere stained with antibodies against CD3, CD4, CD8, CD49b, CD44 andCD62L and analyzed by flow cytometry.

FIG. 44 illustrates that the increased IL-15 levels obtained bystabilization of IL-15 by the IL15Ra are responsible for the increasedbiological effects. The expression levels of IL-15 using all groups ofmice of the experiment shown in FIG. 41 correlate with biologicaleffects. The figure shows the correlation of IL-15 levels with thelevels of NK cells, CD3CD49 cells, and T cells measured in the lung 3days after DNA injection. This indicates that the increased IL-15 levelsobtained by stabilization of IL-15 by the IL15Ra are responsible for theincreased biological effects in a peripheral tissue such as lung.

FIG. 45 illustrates that IL15Ra Stabilizes IL-15. A: IL-15 measurements(ELISA) in extracts and media of cells transfected with IL15tPA6 (IL15t)in the presence or absence of IL-15 receptor-expressing plasmids, IL15Raor IL15sRa. Triplicate samples were measured and bars represent SD ofExtracellular (Extra), Intracellular (Intra) and total IL-15 production.B, C, D: Western blot analyses of IL-15 produced after transfections.Triplicate transfections were loaded on 12% NuPage acrylamide gels. B,cell extracts; C, medium of transfected 293 cells; D is a higherexposure of C to visualize IL15t. Electrophoresed proteins weretransferred to nylon membranes and IL-15 was visualized by polyclonalanti-human IL-15antibody (AF315, R&D, 1:3000 dilution) and an enhancedchemiluminesence assay (ECL).

FIG. 46 illustrates that co-transfection of IL-15 with the full receptoralpha leads to large amounts of cell surface associated IL-15 (complexedwith IL15Ra), whereas cotransfection with the soluble Receptor alphadoes not. Transfected cells were analyzed by flow cytometry aftersurface staining with Phycoerythrin labelled anti-IL-15 Antibody (R&D).The corresponding levels of IL-15 in the media of the transfected 293cells are shown at the table to the right (Quantikine Elisa, R&D).

FIG. 47 illustrates that IL-15 coexpression stabilizes IL15Ra. 293 cellswere transfected with 50 ng of AG79 hIL15Ra or AG98 IL15sRa alone or incombination with AG59 hIL15tPA6 using the Ca phosphate coprecipitationmethod. Cells were harvested after 72 hours; media and cell extractswere analyzed for IL15Ra production by gel electrophoresis (10% NuPAGEgel), and western blot using a goat anti-IL15Ra antibody (1:3000dilution) and a peroxidase-conjugated rabbit anti-goat IgG (1:5000dilution). Full length glycosylated Receptor alpha migrates as a 59 kDaband, whereas the soluble extracellular part of the Receptor alphamigrates as 42 kDa. Sample dilutions of 1:2 and 1:4 were loaded asindicated at the top to quantify the amounts of produced Receptor. Mockindicates 293 cells transfected with control plasmid only (GFP).

FIGS. 48A-D illustrate the N-glycosylation patterns of IL15Ra. FIG. 48A:The predicted structures of IL15Ra and IL15sRa are indicated. Thedifferent domains are indicated. Nglyc indicates potentialN-glycosylation sites. FIGS. 48B-C: Coexpression leads to the productionof more surface full length Receptor and more secretion of IL15sRa inthe medium. Coexpression also releases from cells IL15sRa that is lessglycosylated. These results are consistent with the rapid transport andcleavage of IL15Ra at the surface of the cell in the presence of IL-15.In addition, comparison of the total amounts of IL15Ra producedindicates that in the absence of IL-15 the full length Receptor may alsobe degraded rapidly in the endosomal pathway. In the absence of IL-15,most of the produced IL15sRa from the IL15sRa remains cell associatedand migrates as an ˜28 kDa band, indicating that it is not processed ordegraded post-translationally as rapidly as the full length IL15Ra.Co-expression of IL-15 increased the secreted IL15sRa with concomitantdecrease of the intracellular amount. Cell associated, 1/110 of extractloaded; Media, 1/450 loaded. FIG. 48D is a higher exposure of C tovisualize the low levels of IL15sRa (produced by IL15Ra alone) in themedium. Lanes indicated with (+) contain material treated withN-glycosidase F (NEB) to identify the degree of N-glycosylation of theproduced Receptor.

FIG. 49A illustrates IL-15 production in the plasma of mice injectedwith different DNA expression vectors as indicated. Injection of the wtcDNA expression vector for IL-15 (IL15wt) leads to low level expression,compared to the optimized vector (IL15t, IL15tPA6), which gives an ˜100fold increase in plasma IL-15 in vivo. To measure IL-15 from the wtvector, 1 μg of DNA was injected per mouse in this experiment.Co-injection of mice with the IL15Ra or IL15sRa plasmids resulted in anaddition ˜100 fold increase in plasma IL-15 levels (10⁶-fold totalincrease). Interestingly, whereas the peak production of IL-15 washighest using the construct expressing IL15sRa, plasma levels decreasedmore rapidly. Thus co-injection with full length IL15Ra led to moreprolonged plasma levels of IL-15, consistent with more gradual cleavageand release from the cell surface. ▪ IL-15 wild-type; Δ improved IL-15with tPA6 SIGPRO peptide (IL15t, also called IL15tPA6); ◯ IL15t andwhole IL15Ra; ⋄ IL15t and soluble IL15Ra.

FIG. 49B illustrates improved plasma concentrations of IL-15 whenadministering nucleic acid vectors encoding IL-15 and ILRa at a 1:3ratio (w/w). Mice were injected with 0.2 μg of DNA for each plasmid,except of the group 15+Ra3, which was injected with 0.2 IL-15 plasmidand 0.6 IL15Ra plasmid. Bars indicate SD. Excess of full length Receptorled to prolonged stay of IL-15 in the plasma as indicated by the highlevels at day 3. Thus, coexpression with sRa leads to highest peakvalues of plasma IL-15, whereas coexpression with the full-length Raleads to more prolonged IL-15 levels and possibly function. This ispresumably due to more gradual release of surface IL-15 bound to theReceptor upon cleavage of and production of sRa/IL-15 complexes. Suchcomplexes are bioactive, as indicated by the activity of coexpressedIL-15/sRa, which produced only soluble complexes. Δ improved IL-15 withtPA6 SIGPRO peptide (IL15t); ◯ IL15t and whole IL15Ra (15+Ra);  IL15tand whole IL15Ra at a ratio of 1:3 (w/w) (15+Ra3); ⋄ IL15t and solubleIL15Ra (15+sRa).

FIG. 50 illustrates the size of mesenteric lymph nodes and spleen 3 dayspost DNA injection with the indicated DNAs. GFP DNA expression vectorwas used as negative control. IL-15 expression alone (IL15t) increasedmore dramatically the size of mesenteric lymph nodes compared to thespleen. This may be the result of strong IL15Ra expression in the lymphnodes, which retains plasma IL-15. The levels of plasma IL-15 measuredat 3 days is also indicated.

FIG. 51 illustrates that IL15Ra and IL15sRa are O-glycosylated.Treatment with O-glycosidase (Roche) indicates that the secreted formsof the Receptor alpha are O-glycosylated. Media from 293 cellstransfected with the indicated constructs were treated withO-glycosidase (lanes indicated with +) and compared to the untreatedmaterial (−).

FIG. 52 illustrates increases in lung NK cells 3 days after hydrodynamicDNA delivery of the indicated plasmids in the tail vein of mice.Different groups of mice were injected with 0.1 μg of plasmidsexpressing IL-15tPA6, IL-15tPA6+IL15Ra (full length Receptor alpha),IL-15tPA6+IL15sRa (soluble Receptor alpha). The group indicated withIL15t+Ra.3 received 0.1 μg of IL-15tPA6 and 0.3 μg of IL15Ra plasmids(IL-15 and IL15Ra at a 1:3 ratio (w/w)). This ratio (approximately 1:3)of IL-15 to Receptor DNA showed a trend for more lung NK cells. Thedifference between IL-15 alone and IL-15+sRa is significant (P<0.01,one-way Anova, Dunnett's Multiple Comparison Test).

FIG. 53 illustrates plasma IL-15 concentrations (pg/ml) after injectionof DNA in macaque muscle. Average plasma values of IL-15 measured inmacaque plasma by Elisa (Quantiglo, R&D) at the indicated days. A singleIM injection followed by electroporation using Advisys system(Woodlands, Tex., advisys.net) was performed for each macaque at days 0,14 and 28, as indicated by arrows. Average values for 3 macaquesreceiving the combination of IL-15/15 Receptor alpha (IL15/Ra, circles)or the IL-15 expression vector only (IL15, triangles) are shown. Theresults show that IL15/15Ra vector combination increased dramaticallythe plasma levels of IL-15, whereas IL-15 vector alone did not.

FIG. 54 illustrates that intramuscular injection of IL-15/15Ra DNAvectors leads to increased plasma IL-15 levels. Six Rhesus macaques wereinjected intramascularly in a single site with macaque IL-15/15Ra DNAexpression vectors. Two injections of DNA at days 0 and 14 wereperformed using 100 μg (animals M100, M115, M120) or 250 μg (animalsM122, M125, M126) of each plasmid. DNA (0.5 ml) was electroporated inthe muscle using the Advisys electroporation system under conditions of0.5 Amps, 52 msec pulse length, 80 sec lag time using a constant currentpulse pattern. The results show elevated plasma IL-15 levels in 4/6macaque during the first inoculation, and in 6/6 macaques during thesecond.

FIG. 55 illustrates IL-15 plasma ELISA at days 4, 5, 19 and 20 after twoimmunizations. Concentrations of IL-15 (pg/ml) were measured in macaqueplasma after DNA vaccination together with IL-15/15Ra. Five macaques(M529, M531, M579, M581, M583) were electroporated at days 0 and 15, andplasma was obtained and analyzed by IL-15 ELISA at days 4, 5, 19 and 20.Three animals in the same study (M530, M573, M575; dashed lines) werenot immunized and used as controls. Four of the five electroporatedanimals showed great increases in plasma IL-15, whereas one animal(529M) did not.

FIG. 56 illustrates IL15/15Ra augmented the specific immune responsesagainst SIV, and assisted in the generation of multifunctionalantigen-specific cytokine producing cells (IFNgamma and IL-2) and ofeffector cells. (Top 3 panels): IFNgamma producing cells per millionlymphocytes upon in vitro stimulation with peptide pools for gag, env,nef pol and tat, respectively. The three macaques were vaccinated with amixture of DNA vectors encoding for SIV antigens, IL-15 and IL15Ra atweeks 0, 4, 8, and PBMC were isolated and tested every 2 weeks asindicated. (Bottom panel): SIV specific IL-2 producing T cells permillion lymphocytes at weeks 11-21 (two weeks after release fromtherapy). PBMC were isolated and stimulated in vitro with peptide poolscorresponding to gag, env, nef, tat or pol proteins of SIVmac239. Week11 was the first time that multifunctional IL-2 secreting SIV specificcells were detected in these macaques. These animal participated in aprevious immunotherapy experiment, but did not previously have IL-2producing cells.

FIG. 57 illustrates the presence of circulating multifunctional centralmemory (CM) and effector memory (EM) cells in the DNA vaccinatedmacaques 2 weeks after the third vaccination. CM cells were defined asCD28+CD45RA−. EM cells were CD28-CD45RAlow/+.

FIG. 58 illustrates a map of a construct that coordinately expressesIL-15 and IL15Ra.

FIG. 59 illustrates a map of a construct that coordinately expressesIL-15tPA6 and IL15Ra.

FIG. 60 illustrates a map of a construct that coordinately expressesIL-15tPA6 and IL15sRa.

DETAILED DESCRIPTION 1. Introduction

The cytokine interleukin-15, in encoding nucleic acid or protein form,finds use as an immune cell stimulant (e.g., lymphocyte expansion andactivation) and vaccine adjuvant. Native IL-15 coding sequences do notexpress IL-15 optimally because of several different reasons, includingsignals within the RNA sequence such as potential splice sites and lowstability determinants (oftentimes A/T or A/U rich) sequences embeddedwithin the coding sequences. By minimizing potential splice sites andlow stability sequences from IL-15 sequences, expression of IL-15protein can be increased as much as 4-fold, 5-fold, 6-fold, 8-fold,10-fold, 15-fold, 20-fold, 30-fold or more in comparison to expressionfrom native mammalian IL-15 sequences. A general method has beenestablished for this purpose, comprising changing several codons of theencoded mRNA to alternative codons encoding the same amino acid (see,e.g., U.S. Pat. Nos. 5,965,726; 5,972,596; 6,174,666; 6,291,664;6,414,132; and 6,794,498, the disclosures of each of which are herebyincorporated herein by reference in their entirety for all purposes).This results in the change of any negatively acting signals embeddedinto the RNA without altering the produced protein.

Production of IL-15 protein in mammalian cells can be further increasedby swapping the native IL-15 signal peptide and/or propeptide sequenceswith the signal peptide and/or propeptide sequences from a heterologousprotein, including for example, tissue plasminogen activator, growthhormone or an immunoglobulin protein. Using an improved coding sequencefor mature IL-15 fused to a heterologous signal peptide and/orpropeptide, expression levels of IL-15 mammalian cells can be increased20-fold, 40-fold, 50-fold, 70-fold, 90-fold for more in comparison toexpression from a wild-type IL-15 sequence, and an additional 2-fold,3-fold, 4-fold, 5-fold or more in comparison to expression from animproved IL-15 coding sequence having native signal peptide and/orpropeptide sequences (see, FIG. 1).

2. Nucleic Acid Sequences

The improved high expressing IL-15 nucleic acid sequences of theinvention are usually based on a native mammalian interleukin-15 codingsequence as a template. Nucleic acids sequences encoding nativeinterleukin-15 can be readily found in publicly available databasesincluding nucleotide, protein and scientific databases available on theworldwide web through the National Center for Biotechnology Informationat ncbi.nlm.nih.gov. Native IL-15 nucleic acid sequences can beconveniently cloned from numerous mammalian tissues, including placenta,skeletal muscle, kidney, lung, heart and monocytes/macrophages (see,Grabstein, et al., Science (1994) 264:965). Protocols for isolation andstimulation of desired immune cell populations are well known in theart. See, for example, Current Protocols in Immunology, Coligan, et al.,eds., 1991-2006, John Wiley & Sons.

The sequences are modified according to methods that simultaneouslyrectify several factors affecting mRNA traffic, stability andexpression. Codons are altered to change the overall mRNAAT(AU)-content, to minimize or remove all potential splice sites, and toalter any other inhibitory sequences and signals affecting the stabilityand processing of mRNA such as runs of A or T/U nucleotides, AATAAA,ATTTA and closely related variant sequences, known to negatively affectmRNA stability. The methods applied to IL-15 coding nucleic acidsequences in the present application have been described in U.S. Pat.Nos. 6,794,498; 6,414,132; 6,291,664; 5,972,596; and 5,965,726 thedisclosures of each of which are hereby incorporated herein by referencein their entirety for all purposes.

Generally, the changes to the nucleotide bases or codons of a codingIL-15 sequence do not alter the amino acid sequence comprising an IL-15protein from the native IL-15 protein. The changes are based upon thedegeneracy of the genetic code, utilizing an alternative codon for anidentical amino acid, as summarized in Table 1, above. In certainembodiments, it will be desirable to alter one or more codons to encodea similar amino acid residue rather than an identical amino acidresidue. Applicable conservative substitutions of coded amino acidresidues are described above.

Oftentimes, in carrying out the present methods for increasing thestability of an IL-15 coding sequence, a relatively more A/T-rich codonof a particular amino acid is replaced with a relatively more G/C richcodon encoding the same amino acid (see, for example FIGS. 2 and 4). Forexample, amino acids encoded by relatively more A/T-rich and relativelymore G/C rich codons are shown in Table 2.

TABLE 2 relatively more relatively more Amino Acid A/T-rich codon(s)G/C-rich codon(s) Ala GCA, GCT GCC, GCG Asn AAT AAC Asp GAT GAC Arg CGA,CGT, AGA CGC, CGG, AGG Cys TGT TGC Gln CAA CAG Glu GAA GAG Gly GGA, GGTGGC, GGG His CAT CAC Ile ATA, ATT ATC Leu TTA, CTA, CTT TTG, CTC, CTGLys AAA AAG Phe TTT TTC Pro CCA, CCT CCC, CCG Ser TCA, TCT, AGT TCC,TCG, AGC Thr ACA, ACT ACC, ACG Tyr TAT TAC Val GTA, GTT GTC, GTG

Depending on the number of changes introduced, the improved IL-15 and/orIL15Ra nucleic acid sequences of the present invention can beconveniently made as completely synthetic sequences. Techniques forconstructing synthetic nucleic acid sequences encoding a protein orsynthetic gene sequences are well known in the art. Synthetic genesequences can be commercially purchased through any of a number ofservice companies, including DNA 2.0 (Menlo Park, Calif.), Geneart(Toronto, Ontario, Canada), CODA Genomics (Irvine, Calif.), andGenScript, Corporation (Piscataway, N.J.). Alternatively, codon changescan be introduced using techniques well known in the art. Themodifications also can be carried out, for example, by site-specific invitro mutagenesis or by PCR or by any other genetic engineering methodsknown in art which are suitable for specifically changing a nucleic acidsequence. In vitro mutagenesis protocols are described, for example, inIn Vitro Mutagenesis Protocols, Braman, ed., 2002, Humana Press, and inSankaranarayanan, Protocols in Mutagenesis, 2001, Elsevier Science Ltd.

High level expressing improved IL-15 and/or IL15Ra sequences can beconstructed by altering select codons throughout a native IL-15 and/orIL15Ra nucleic acid sequence, or by altering codons at the 5′-end, the3′-end, or within a middle subsequence. It is not necessary that everycodon be altered, but that a sufficient number of codons are altered sothat the expression (i.e., transcription and/or translation) of theimproved IL-15 and/or IL15Ra nucleic acid sequence is at least about10%, 25%, 50%, 75%, 1-fold, 2-fold, 4-fold, 8-fold, 20-fold, 40-fold,80-fold or more abundant in comparison to expression from a native IL-15and/or IL15Ra nucleic acid sequence under the same conditions.Expression can be detected over time or at a designated endpoint, usingtechniques known to those in the art, for example, using gelelectrophoresis or anti-IL-15 or anti-IL15Ra antibodies in solutionphase or solid phase binding reactions (e.g., ELISA,immunohistochemistry). Interleukin-15 ELISA detection kits arecommercially available from, for example, RayBiotech, Norcross, Ga.;Antigenix America, Huntington Station, N.Y.; eBioscience, San Diego,Calif.; Biosource (Invitrogen), Camarillo, Calif.; R & D Systems(Minneapolis, Minn.), and PeproTech, Rocky Hill, N.J.

Usually at least about 50% of the changed nucleotides or codons whosepositions are identified in FIG. 8 are changed to another nucleotide orcodon such that the same or a similar amino acid residue is encoded. Inother embodiments, at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 95%, 97%, 98%, 99% of the changed codons identified in FIG. 8 arechanged to another nucleotide or codon such that the same or a similaramino acid residue is encoded.

The nucleotide positions that can be changed for an improved IL-15nucleic acid sequence as identified in FIG. 8 are 6, 9, 15, 18, 21, 22,27, 30, 33, 49, 54, 55, 57, 60, 63, 69, 72, 75, 78, 81, 84, 87, 90, 93,96, 105, 106, 114, 120, 123, 129, 132, 135, 138, 141, 156, 159, 162,165, 168, 169, 174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207,210, 213, 216, 217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261,277, 283, 285, 291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324,327, 330, 333, 336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387,390, 393, 396, 402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450,453, 456, 459, 462, 468, 483 and 486.

The GC-content of an improved IL-15 nucleic acid sequence is usuallyincreased in comparison to a native IL-15 nucleic acid sequence whenapplying the present methods. For example, the GC-content of an improvedIL-15 nucleic acid sequence can be at least about 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% or more.

In some embodiments, the native IL-15 signal peptide (SIG) sequence orsignal peptide and propeptide (SIG-PRO) sequence is replaced with thesecretory SIG sequence or SIG-PRO sequence from a heterologous protein(i.e., a protein other than IL-15) (see, for example, FIG. 9).Exemplified signal peptide and propeptide sequences include those fromtissue plasminogen activator (tPA) protein, growth hormone, GM-CSF, andimmunoglobulin proteins. Tissue plasminogen activator signal peptide andpropeptide sequences are known in the art (see, Delogu, et al, InfectImmun (2002) 70:292; GenBank Accession No. E08757). Growth hormonesignal peptide and propeptide sequences also are known in the art (see,Pecceu, et al., Gene (1991) 97:253; GenBank Accession Nos. M35049 andX02891). Immunoglobulin signal peptide and propeptide sequences, forexample of immunoglobulin heavy chains, also are known in the art (see,Lo, et al., Protein Eng. (1998) 11:495 and Gen Bank Accession Nos.Z75389 and D14633). Signal peptide-IL-15 fusion proteins andSIG-PRO-IL-15 fusion proteins can have cleavage sequences recognized bysite-specific proteases incorporated at one or more sites of the fusionproteins, for example, immediately before the N-terminal amino acidresidue of the mature IL-15. Numerous cleavage sequences recognized bysite-specific proteases are known in the art, including those for furin,thrombin, enterokinase, Factor Xa, and the like.

In one embodiment, the native IL-15 signal peptide and propeptidesequences are replaced with the signal peptide and propeptide sequencesfrom tPA. In a further embodiment, the tPA SIG-PRO sequence is alteredto remove one or more amino acid residues and/or to incorporate aprotease cleavage site (e.g., thrombin, enterokinase, Factor Xa). See,FIG. 12.

In some embodiments, the native IL15Ra signal peptide (SIG) sequence orsignal peptide and propeptide (SIG-PRO) sequence is replaced with thesecretory SIG sequence or SIG-PRO sequence from a heterologous protein(i.e., a protein other than IL15Ra). Exemplified signal peptide andpropeptide sequences include those discussed above, for example, tissueplasminogen activator (tPA) protein, GM-CSF, growth hormone, andimmunoglobulin proteins. In some embodiments, the IL15Ra nucleicsequences do not encode an immunoglobulin sequence, for example, anoperably linked Fc sequence.

Once a high level expressing improved IL-15 nucleic acid sequence hasbeen constructed, it can be cloned into a cloning vector, for example aTA-cloning® vector (Invitrogen, Carlsbad, Calif.) before subjecting tofurther manipulations for insertion into one or more expression vectors.Manipulations of improved IL-15 nucleic acid sequences, includingrecombinant modifications and purification, can be carried out usingprocedures well known in the art. Such procedures have been published,for example, in Sambrook and Russell, Molecular Cloning: A LaboratoryManual, 2000, Cold Spring Harbor Laboratory Press and Current Protocolsin Molecular Biology, Ausubel, et al., eds., 1987-2006, John Wiley &Sons.

3. Expression Vectors

IL-15 and IL15Ra sequences can be recombinantly expressed from anexpression vector containing an improved IL-15 and/or IL15Ra codingsequence. One or both of the IL-15 and/or IL15Ra coding sequences can beimproved. The expression vectors of the invention have an expressioncassette that will express one or both of IL-15 and IL15Ra in amammalian cell. The IL-15 and IL15Ra can be expressed from the same ormultiple vectors. The IL-15 and IL15Ra can be expressed from the samevector from one or multiple expression cassettes (e.g., a singleexpression cassette with an internal ribosome entry site; or a doubleexpression cassette using two promoters and two polyA sites). Withineach expression cassette, sequences encoding an IL-15 and an IL15Ra willbe operably linked to expression regulating sequences. “Operably linked”sequences include both expression control sequences that are contiguouswith the nucleic acid of interest and expression control sequences thatact in trans or at a distance to control the gene of interest.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that promote RNAexport (e.g., a constitutive transport element (CTE), a RNA transportelement (RTE), or combinations thereof, including RTEm26CTE); sequencesthat enhance translation efficiency (e.g., Kozak consensus sequence);sequences that enhance protein stability; and when desired, sequencesthat enhance protein secretion.

The expression vector can optionally also have a third independentexpression vector for expressing a selectable marker. Selectable markersare well known in the art, and can include, for example, proteins thatconfer resistance to an antibiotics, fluorescent proteins, antibodyepitopes, etc. Exemplified markers that confer antibiotic resistanceinclude sequences encoding β-lactamases (against β-lactams includingpenicillin, ampicillin, carbenicillin), or sequences encoding resistanceto tetracylines, aminoglycosides (e.g., kanamycin, neomycin), etc.Exemplified fluorescent proteins include green fluorescent protein,yellow fluorescent protein and red fluorescent protein.

The promoter(s) included in the expression cassette(s) should promoteexpression of the IL-15 and/or an IL15Ra polypeptide in a mammaliancell. The promoter or promoters can be viral, oncoviral or nativemammalian, constitutive or inducible, or can preferentially regulatetranscription of IL-15 and/or IL15Ra in a particular tissue type or celltype (e.g., “tissue-specific”).

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. Exemplified constitutivepromoters in mammalian cells include oncoviral promoters (e.g., simiancytomegalovirus (CMV), human CMV, simian virus 40 (SV40), rous sarcomavirus (RSV)), promoters for immunoglobulin elements (e.g., IgH),promoters for “housekeeping” genes (e.g., β-actin, dihydrofolatereductase).

In another embodiment, inducible promoters may be desired. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. Inducible promoters are those which areregulated by exogenously supplied compounds, including withoutlimitation, a zinc-inducible metallothionine (MT) promoter; an isopropylthiogalactose (IPTG)-inducible promoter, a dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter; a tetracycline-repressiblesystem (Gossen et al, Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992));the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech.,15: 239-243 (1997) and Wang et al., Gene Ther., 4: 432-441 (1997)); andthe rapamycin-inducible system (Magari et al. J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters which can beuseful in this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, or in replicatingcells only.

In another embodiment, the native promoter for a mammalian IL-15 can beused. The native promoter may be preferred when it is desired thatexpression of improved IL-15 sequences should mimic the nativeexpression. The native promoter can be used when expression of theimproved IL-15 and/or IL15Ra must be regulated temporally ordevelopmentally, or in a tissue-specific manner, or in response tospecific transcriptional stimuli. In a further embodiment, other nativeexpression control elements, such as enhancer elements, polyadenylationsites or Kozak consensus sequences may also be used to mimic expressionof native IL-15 and/or IL15Ra.

In another embodiment, the improved IL-15 and/or IL15Ra sequences can beoperably linked to a tissue-specific promoter. For instance, ifexpression in lymphocytes or monocytes is desired, a promoter active inlymphocytes or monocytes, respectively, should be used. Examples ofpromoters that are tissue-specific are known for numerous tissues,including liver (albumin, Miyatake et al. J. Virol., 71: 5124-32 (1997);hepatitis B virus core promoter, Sandig et al., Gene Ther., 3: 1002-9(1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther. 7:1503-14 (1996)), bone (osteocalcin, Stein et al., Mol. Biol. Rep., 24:185-96 (1997); bone sialoprotein, Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor α chain),neuronal (neuron-specific enolase (NSE) promoter, Andersen et al. Cell.Mol. Neurobiol., 13: 503-15 (1993); neurofilament light-chain gene,Piccioli et al., Proc. Natl. Acad. Sci. USA, 88: 5611-5 (1991); theneuron-specific vgf gene, Piccioli et al., Neuron, 15: 373-84 (1995));among others.

In some embodiments, the improved IL-15 and/or IL15Ra sequences areoperably linked to one or more mRNA export sequences. Exemplified mRNAexport elements include the constitutive transport element (CTE), whichis important for the nucleo-cytoplasmic export of the unspliced RNA ofthe simian type D retroviruses. Another exemplified RNA export elementincludes the RNA transport element (RTE), which is present in a subsetof rodent intracisternal A particle retroelements. The CTE and RTEelements can be used individually or in combination. In one embodiment,the RTE is an RTEm26 (e.g., SEQ ID NO:22). In one embodiment, the RTEM26and the CTE are positioned in the 3′-untranslated region of a transcriptencoded by the expression cassette. Often, the RTE and the CTE areseparated by 100 nucleotides or less. In some embodiments, the RTE andthe CTE are separated by 30 nucleotides or less. In one embodiment, theRTE and the CTE are comprised by the sequence set forth in SEQ ID NO:23(RTEm26CTE). RNA transport elements for use in further increasing theexpression of improved IL-15 sequences are described, for example, inInternational Patent Publication No. WO 04/113547, the disclosure ofwhich is hereby incorporated herein by reference in its entirety for allpurposes.

4. Mammalian Cells

The expression vectors of the invention can be expressed in mammalianhost cells. The host cells can be in vivo in a host or in vitro. Forexample, expression vectors containing high-level expressing IL-15and/or IL15Ra nucleic acid sequences can be transfected into culturedmammalian host cells in vitro, or delivered to a mammalian host cell ina mammalian host in vivo.

Exemplary host cells that can be used to express improved IL-15 and/orIL15Ra nucleic acid sequences include mammalian primary cells andestablished mammalian cell lines, including COS, CHO, HeLa, NIH3T3, HEK293-T, RD and PC12 cells. Mammalian host cells for expression of IL-15and/or IL15Ra proteins from high level expressing improved IL-15 and/orIL15Ra nucleic acid sequences are commercially available from, forexample, the American Type Tissue Collection (ATCC), Manassas, Va.Protocols for in vitro culture of mammalian cells is also well known inthe art. See, for example, Handbook of Industrial Cell Culture:Mammalian, Microbial, and Plant Cells, Vinci, et al., eds., 2003, HumanaPress; and Mammalian Cell Culture: Essential Techniques, Doyle andGriffiths, eds., 1997, John Wiley & Sons.

Protocols for transfecting mammalian host cells in vitro and expressingrecombinant nucleic acid sequences are well known in the art. See, forexample, Sambrook and Russell, and Ausubel, et al, supra; Gene Deliveryto Mammalian Cells: Nonviral Gene Transfer Techniques, Methods inMolecular Biology series, Heiser, ed., 2003, Humana Press; and Makrides,Gene Transfer and Expression in Mammalian Cells, New ComprehensiveBiochemistry series, 2003, Elsevier Science. Mammalian host cellsmodified to express the improved IL-15 nucleic acid sequences can betransiently or stably transfected with a recombinant vector. Theimproved IL-15 and/or IL15Ra sequences can remain epigenetic or becomechromosomally integrated.

5. Administration of Improved IL-15 and/or IL15Ra Sequences

The high level expression improved IL-15 and/or IL15Ra nucleic acidsequences are suitable for administration to an individual alone, forexample to treat immunodeficiency (e.g., promote the expansion oflymphocytes, including B cells, T cells, NK cells and NK T cells), or asan adjuvant co-delivered with one or more vaccine antigens. The use ofIL-15 and/or IL15Ra for the treatment of immune deficiency and as anadjuvant is known in the art (see, for example, Diab, et al., supra;Ahmad, et al, supra; and Alpdogan and van den Brink, supra).

In one embodiment, high level expressing improved IL-15 and/or IL15Ranucleic acid sequences are co-administered with one or more vaccineantigens, with at least the improved IL-15 and/or IL15Ra nucleic acidsequences delivered as naked DNA. The one or more antigen can bedelivered as one or more polypeptide antigens or a nucleic acid encodingone or more antigens. Naked DNA vaccines are generally known in the art;see, Wolff, et al., Science (1990) 247:1465; Brower, NatureBiotechnology (1998) 16:1304; and Wolff, et al., Adv Genet (2005) 54:3.Methods for the use of nucleic acids as DNA vaccines are well known toone of ordinary skill in the art. See, DNA Vaccines, Ertl, ed., 2003,Kluwer Academic Pub and DNA Vaccines: Methods and Protocols, Lowrie andWhalen, eds., 1999, Humana Press. The methods include placing a nucleicacid encoding one or more antigens under the control of a promoter forexpression in a patient. Co-administering high level expressing improvedIL-15 and/or IL15Ra nucleic acid sequences further enhances the immuneresponse against the one or more antigens. Without being bound bytheory, following expression of the polypeptide encoded by the DNAvaccine, cytotoxic T-cells, helper T-cells and antibodies are inducedwhich recognize and destroy or eliminate cells or pathogens expressingthe antigen.

In one embodiment, one or both of the IL-15 and/or IL15Ra sequences areco-administered as proteins.

The invention contemplates compositions comprising improved IL-15 and/orIL15Ra amino acid and nucleic acid sequences in a physiologicallyacceptable carrier. While any suitable carrier known to those ofordinary skill in the art may be employed in the pharmaceuticalcompositions of this invention, the type of carrier will vary dependingon the mode of administration. For parenteral administration, includingintranasal, intradermal, subcutaneous or intramuscular injection orelectroporation, the carrier preferably comprises water, saline, andoptionally an alcohol, a fat, a polymer, a wax, one or more stabilizingamino acids or a buffer. General formulation technologies are known tothose of skill in the art (see, for example, Remington: The Science andPractice of Pharmacy (20th edition), Gennaro, ed., 2000, LippincottWilliams & Wilkins; Injectable Dispersed Systems: Formulation,Processing And Performance, Burgess, ed., 2005, CRC Press; andPharmaceutical Formulation Development of Peptides and Proteins, Frkjret al., eds., 2000, Taylor & Francis).

Naked DNA can be delivered in solution (e.g., a phosphate-bufferedsaline solution) by injection, usually by an intra-arterial,intravenous, subcutaneous or intramuscular route. In general, the doseof a naked nucleic acid composition is from about 10 μg to 10 mg for atypical 70 kilogram patient. Subcutaneous or intramuscular doses fornaked nucleic acid (typically DNA encoding a fusion protein) will rangefrom 0.1 mg to 50 mg for a 70 kg patient in generally good health.

DNA vaccinations can be administered once or multiple times. In someembodiments, the improved IL-15 and/or IL15Ra nucleic acid sequences areadministered more than once, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15,20 or more times as needed to induce the desired response (e.g.,specific antigenic response or proliferation of immune cells). Multipleadministrations can be administered, for example, bi-weekly, weekly,bi-monthly, monthly, or more or less often, as needed, for a time periodsufficient to achieve the desired response.

In some embodiments, the improved IL-15 and/or IL15Ra nucleic acidcompositions are administered by liposome-based methods, electroporationor biolistic particle acceleration. A delivery apparatus (e.g., a “genegun”) for delivering DNA into cells in vivo can be used. Such anapparatus is commercially available (e.g., BioRad, Hercules, Calif.,Chiron Vaccines, Emeryville, Calif.). Naked DNA can also be introducedinto cells by complexing the DNA to a cation, such as polylysine, whichis coupled to a ligand for a cell-surface receptor (see, for example,Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson et al.(1992) J. Biol. Chem. 267:963-967; and U.S. Pat. Nos. 5,166,320;6,846,809; 6,733,777; 6,720,001; 6,290,987). Liposome formulations fordelivery of naked DNA to mammalian host cells are commercially availablefrom, for example, Encapsula NanoSciences, Nashville, Tenn. Anelectroporation apparatus for use in delivery of naked DNA to mammalianhost cells is commercially available from, for example, InovioBiomedical Corporation, San Diego, Calif.

The improved IL-15 and/or IL15Ra nucleic acid vaccine compositions areadministered to a mammalian host. The mammalian host usually is a humanor a primate. In some embodiments, the mammalian host can be a domesticanimal, for example, canine, feline, lagomorpha, rodentia, rattus,hamster, murine. In other embodiment, the mammalian host is anagricultural animal, for example, bovine, ovine, porcine, equine, etc.

6. Methods of Expressing IL-15 and/or IL15Ra in Mammalian Cells

The methods of the present invention provide for expressing IL-15 and/orIL15Ra in a mammalian cell by introducing a recombinant vector into thecell to express the high level improved IL-15 and/or IL15Ra nucleic acidsequences described herein. The modified mammalian cell can be in vitroor in vivo in a mammalian host.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

The strategy for introducing nucleotide changes into IL-15 sequences isto simultaneously rectify several factors affecting mRNA traffic,stability and expression. Codons are altered to change the overall mRNAAT(AU)-content or to remove any other inhibitory signals within the RNAsuch as all potential splice sites (computer programs predictingpotential splice sites can be found for example at web sites such asfruitfly.org/seq_tools/splice.html, or sun1.softberry.com/berry.phtml)and also to alter sequences such as runs of A or T/U nucleotides,AATAAA, ATTTA and closely related variant sequences, known to negativelyaffect mRNA. By substituting codons with a different codon encoding theidentical amino acid, the chosen codon can be more GC-rich, or can havea different sequence that is sufficient to alter the RNA structure. Thisapproach has been described in several patents, each of which is herebyincorporated herein by reference in their entirety: U.S. Pat. Nos.5,965,726; 5,972,596; 6,174,666; 6,291,664; 6,414,132; and 6,794,498.

Procedures

Standard lab techniques are used to generate, purify and sequenceplasmid DNAs. One microgram (1 μg) of the plasmids containing theindicated IL-15 coding sequence were transfected into human 293 or RDcells seeded into 60 mm plates the day before with 10⁶ cells usingcalcium coprecipitation technique (293 cells) and the SuperFect Reagentprotocol (Qiagen) for RD4 cells. 2-3 days later, intracellular andextracellular and total IL-15 protein was measured using commercial kits(R&D system). Due to the high homology of the human and Rhesus IL-15proteins, their protein levels were determined by the same commercialELISA kit. The results of different experiments are shown in FIGS. 7,10, 11, 13, 25, 26 and 27.

Example 2

This example demonstrates the improved expression sequences for IL-15Receptor alpha and the soluble (extracellular) part of IL-15 Receptoralpha (IL15sRa). These improved sequences increased protein expressionof the IL-15 Receptor alpha and provide a method to further optimize theactivity of IL-15 in vivo and in vitro.

Results

FIGS. 39 and 40 show that co-expression of IL-15 with IL-15 Receptoralpha optimized sequences in human 293 cells in vitro using standardtransfection methods led to a dramatic increase of total IL-15 levelsmeasured. This increase is the result of stabilization of the IL-15molecule by binding to the whole IL-15 receptor alpha or to theextracellular part of the IL-15 receptor alpha. The results were similarif the IL-15 and the receptor were expressed by two different plasmidsor expressed by a single plasmid from two different promoters.

FIG. 41 shows a great increase in the levels of lung NK cells and alsoincreases of Lung CD3+CD49+ cells when IL-15 and IL-15 receptor DNA weredelivered and expressed in mice tissues after tail vein injection. Thenumber of cells is given per 10⁵ cells in the analysis file.

FIG. 42 shows the increase of NK cells and T cells in the liver of miceafter DNA injection of IL-15 alone, IL-15+IL-15 Receptor alpha, orIL-15+IL-15 soluble Receptor alpha. The number of cells is given per 10⁶cells in the analysis file.

FIG. 43 shows the increase in the effector cells in the spleen (totaleffectors and CD8 effectors, respectively). The lack of CD62L defines apopulation of murine memory T cells with effector phenotype.

FIG. 44 indicates that the increased IL-15 levels obtained bystabilization of IL-15 by the IL15Ra are responsible for the increasedbiological effects.

Methods: Expression in Cultured Cells

Human 293 cells were transfected with 0.1 μg of the human IL15tPA6OPTplasmid either alone or together with 0.1 μg of a plasmid expressing theRNA optimized versions of the human IL-15 receptor alpha using eitherthe full length form (huIL15RaOPT) or the soluble form (hu sIL15RaOPT).Medium was taken at 24 and at 48 hours posttransfection and cells wereharvested at 48 hrs. IL-15 levels were measured using Quantikine HumanIL-15 immunoassay (R&D systems) prior to release from the cell.

Expression in Mouse

Six week old Balb/c mice were either injected with DNA via theintramuscular route into both of the quadriceps or hydrodynamically viathe tail vein. For the hydrodynamic DNA delivery, the mice were injectedwith 1 μg of human IL15-tPA6OPT plasmid either alone or together with 1μg the plasmid expressing the human IL-15 Receptor alpha using eitherthe intact form (huIL15RaOPT) or the soluble form (hu sIL15RaOPT) in 1.6ml of sterile 0.9% NaCl via the tail vein. Three days later, mice weresacrificed and the levels of IL-15 were measured in the plasma using acommercial chemiluminescent immunoassay (Quantiglo, R&D). Thebioactivity of IL-15 was measured in liver, spleen and lung usingmulticolor FACS. Briefly, cells were staining ex-vivo with the followingpanel of conjugated rat anti-mouse antibodies: APCCy7-CD3, PerCP-CD4,PECy7- CD8, APC-CD44, FITC-CD49b and PE-CD62L, BD-Pharmingen andanalyzed by flow cytometry. Murine NK cells are phenotypicallyidentified as CD3-CD49b+.

Example 3

This example demonstrates the mutual stabilization of IL-15 and IL-15Receptor alpha. The data demonstrate that combined production of IL-15and IL15Ra endogenously allows the two molecules to efficiently combinein a functional secreted form.

In the presence of IL-15, the IL-15 Receptor alpha is rapidly deliveredto the surface of the cell (see, FIG. 46) and it is also rapidly cleaved(see, FIG. 47). Thus, expression of the full receptor leads rapidly tothe soluble receptor/IL-15 complex, which is released in the circulationand can act at distant tissues.

This example follows the in vivo production of IL-15 by measuring theplasma levels over time (see, FIG. 49). The soluble receptor/IL-15 genecombination gives a sharp peak of plasma IL-15, which is rapidlydecreased, whereas the complete receptor/IL-15 combination gives a lowerpeak but decays less rapidly. This allows the delivery of differentformulations having more or less prolonged action in vivo.

Results

Cells transfected with IL-15 alone express and secrete IL-15inefficiently. In addition, like many cytokine mRNAs, the IL-15 mRNA isunstable and can be improved by RNA/codon optimization. RNA/codonoptimization can be used to increase IL-15 and IL15Ra mRNA levels andexpression. In addition, the secretory pre-peptide of IL-15 can beexchanged with the tissue Plasminogen Activator (tPA) secretory leaderpeptide, or with other secretory peptides such as IgE or GM-CSF. Theseimprovements have resulted in a 100-fold increase of expression usingthe human CMV promoter and Bovine Growth Hormone polyadenylation signalin standard expression vectors.

FIG. 45 shows the in vitro expression of IL-15 after transfection inhuman 293 cells. The use of optimized expression vector (IL15t, whichindicates IL15tPA6OPT) having the tissue Plasminogen Activator (tPA)prepro leader sequence produced easily detectable levels of IL-15 in themedia (i.e., extracellularly and intracellularly). Furthermore,co-expression of IL-15 together with IL15Ra resulted in a dramaticincrease of IL-15 production, both intracellularly and extracellularly.This expression level was approximately 20-fold higher compared to theexpression from the wild type cDNA.

Coexpression of IL-15 with the full length (i.e., whole) IL15Ra resultedin high levels of the IL-15 and IL15Ra molecules localized in the cellsurface of expressing cells (FIG. 46), whereas coexpression of IL-15with the soluble, extracellular portion of IL15Ra (i.e., soluble IL-15)resulted in rapid secretion of the complex in the medium. The totalincrease in IL-15 steady-state levels was 4-fold in the presence ofIL15Ra and 7-fold in the presence of IL15sRa, as measured by ELISA (FIG.45A).

Conversely, the presence of co-expressed IL-15 also increased the levelsof IL15Ra and IL15sRa (FIG. 47). Western blot analysis using differentdilutions of media and cell extracts after transfections of 293 cellswith IL15Ra or IL15sRa in the presence or absence of IL-15 showed a 3-to 8-fold increase in receptor steady-state levels in the presence ofIL-15. The receptor increase is in general similar to the IL-15 increaseupon coexpression, measured above.

After expression of the membrane associated full IL15Ra, largequantities of the soluble extracellular portion were detected in themedium, consistent with rapid cleavage of the receptor and generation ofthe soluble form. When IL-15 was co-expressed, the levels of solublereceptor in the medium were elevated (FIG. 47C). Expression of IL15sRaresulted in high levels of a ˜28 kDa intracellular form of the receptor,which is the primary transcript of the transfected cDNA, without anyglycosylation, as well as an additional N-glycosylated ˜30 kDa form (seebelow). Low levels of the fully glycosylated IL15sRa were foundcell-associated, whereas most of it was secreted in the medium. In thepresence of co-expressed IL-15, the intracellular non-glycosylated formwas drastically reduced, whereas the glycosylated forms, especially theextracellular, were greatly increased. These results are consistent withthe conclusion that an early intracellular association of IL-15 to itsreceptor alpha takes place during the production and secretion of thesetwo molecules. In the absence of IL-15, IL15sRa remains to a largeextent intracellular and it is not processed or secreted rapidly.

Both IL-15 and IL15Ra are glycosylated molecules and migrate as multiplebands in SDS-PAGE gels. IL15Ra is both N- and O-glycosylated (Dubois etal., 1999 J Biol Chem 274(38):26978-84), whereas IL-15 isN-glycosylated. It has been reported that the different IL15Ra proteinproducts are due to alternate N- and O-glycosylations of a 39-kDaprecursor (Dubois et al., 1999). Treatment with N- or O-glycosidasesrevealed that most of the cell associated IL15Ra receptor is rapidlyglycosylated. In contrast, expression of the IL15sRa alone revealed anapproximately 28 kDa band for the IL15sRa, which was only seenintracellularly. In the presence of IL15, this intracellular banddecreased dramatically with coordinate increase in the extracellularglycosylated forms.

To determine whether the increased expression resulted in betterbiological activity, IL-15 and IL15Ra or IL15sRa DNA molecules wereexpressed in mice after hydrodynamic DNA delivery by tail veininjection. Mice were administered 0.1 μg to 2 μg DNA for theseexperiments, and IL-15 levels in the plasma were measured. Three daysafter a single DNA injection, mice were sacrificed and selected tissueswere analyzed for the number and phenotype of T cells, NK cells, andother lymphocyte subsets by flow cytometry. FIG. 52 shows thatco-expression of IL-15 and the Receptor alpha increased the number of NKcells in the lung. This increase was more prominent when the plasmidexpressing the receptor was injected at a higher molar ratio (3:1, 0.1μg of IL-15 plasmid and 0.3 μg of IL15Ra plasmid). Co-expression of thesoluble part of IL-15 Receptor alpha gave a dramatic increase in lung NKcells.

Example 4

This example shows the use of IL-15/IL15Ra combination in a therapeuticvaccination of macaques. The IL-15/IL15Ra combination increased antigenspecific cells, especially CD8 effectors, and also cells that expressIL-2 or IL-2 and IFNgamma upon antigen stimulation (i.e.,multifunctional cells, which are considered important for effectivevaccination).

This example also follows expression of IL-15 in macaque plasma, andshow that IL-15/15Ra co-expression achieves detectable production inmacaque plasma. Control experiments show that this production is muchhigher compared to animals receiving only IL-15 DNA.

Three macaques were subjected to a second round of antiretroviraltreatment (“ART”) and DNA vaccination using plasmids expressing improvedIL-15 and IL-15 Receptor alpha (IL15Ra) Immunization was done byelectroporation using the following plasmid mix: Two injections of 0.5ml were performed for each animal. Peripheral blood monocytes (“PBMC”)were isolated at 2 week intervals and analyzed for numbers ofSIV-specific cells using 10 parameter flow cytometry. This allowed theenumeration and phenotypic analysis of lymphocytes producing IFNg, IL-2or TNFa in response to stimulation by peptide pools corresponding togag, pol, env, nef, and tat proteins of SIVmac259.

The results of this analysis (FIG. 56) show a dramatic increase ofaverage and peak responses of SIV-specific cytokine producing cells. Allthree animals had low levels of IFNg producing cells during ART andprior to DNA vaccination. This is expected since ART decreased SIV toundetectable levels in all three animals. Upon vaccination a persistentincrease of SIV-specific cells was detected. More importantly,vaccination generated IL-2-secreting cells (FIG. 56) as well as doubleIFNg and IL-2 secreting cells (i.e., multifunctional cells). Thisoccurred only after the third DNA vaccination, whereas in all previousdeterminations these macaques did not have any polyfunctional cytokinesecreting cells in their peripheral blood.

The three vaccinated macaques showed dramatic increases in the number ofSIV-specific cytokine-producing cells in PBMC with either central memory(CM) or effector memory (EM) phenotype (FIG. 57). The appearance ofincreased levels of effector cells in PBMC upon vaccination with theoptimized mix of DNAs is in contrast to our previous experience, whereDNA vaccination was able to generate SIV-specific central memory but noteffector memory cells. We attribute this to the more optimal mix of DNAvaccines and to the presence of effective levels of IL15/IL15Racytokine.

Macaque administered DNA encoding IL-15 without co-administration of DNAencoding IL15Ra did not have IL-2 producing cells.

In summary, the optimized DNA vaccine vector mix and the inclusion ofoptimized levels of DNAs expressing IL-15 and IL15Ra resulted in adramatic increase in antigen-specific cells detected in the peripheralblood. In addition to increased levels of cells, important phenotypicdifferences were detected by our analysis. The vaccine-generatedantigen-specific cells were shown to include IL-2 producing as well asdual IFNg and IL-2 producing cells. Vaccination with IL-15 and IL15Ragenerated antigen-specific cells having an effector phenotype inaddition to central memory antigen-specific cells. CD8+ effector cellsare expected to be active against virus-infected cells, therefore thesemacaques will be able to better control virus upon release from ART.Surprisingly, approximately 1-2% of circulating lymphocytes are SIVspecific as a result of the dramatic response to DNA vaccination. Thisindicates that DNA vaccination alone under optimized conditions cangenerate a strong, diverse, long-lasting and multifunctional repertoireof antigen specific cells. DNA vaccination was administered successfullymany times (up to a total of 8 times) without adverse effects. Moreover,repeated administrations resulted in the production of multifunctional Tcells. This represents a dramatic improvement in comparison to previousvaccination protocols.

DNA injection of IL15/IL15Ra combination appears responsible for a greatmobilization of effector cells, which are detected in PBMC on their wayto peripheral sites. If this is the case, these results suggest theeffectiveness of optimized IL15/IL15Ra combination as DNA or protein toenhance the mobilization and function of lymphocytes at optimalintervals in vivo. This immunotherapy with IL-15 can be used to enhancethe effects of therapeutic vaccination and can also be used to enhancethe immune response against the virus in the absence of therapeuticvaccination or for a long time after vaccination.

The DNA vaccine vectors used in this therapeutic vaccination were a mixcomposed of six SIV antigen-expressing plasmids and 2 rhesus IL-15/IL-15Receptor alpha expressing plasmids. LAMP-pol and LAMP-NTV plasmidsproduce protein fusions of pol or NefTatVif, respectively, to humanLysosomal Associated Membrane Protein.

2S-CATEgagDX

21S-MCP3p39gag

99S-Env

73S-MCP3-env

103S-LAMP-pol

147S-LAMP-NTV

Rhesus IL-15/IL-15 Receptor Alpha Producing Plasmids:

AG65-rhIL15tPA6

AG120-rhIL15Ra

1.-60. (canceled)
 61. A polynucleotide comprising a nucleic acidsequence encoding an interleukin-15 (IL-15) polypeptide, wherein thenucleic acid sequence has at least 85% sequence identity to nucleotides145-489 SEQ ID NO:3.
 62. The polynucleotide of claim 61, wherein thenucleic acid sequence has non-native nucleic acid bases at 80% or moreof the 80 nucleotide positions 156, 159, 162, 165, 168, 169, 174, 177,180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217, 219,222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291, 294,297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339,351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402, 405,414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462, 468,483 of SEQ ID NO:3.
 63. The polynucleotide of claim 61, wherein thenucleic acid sequence has non-native nucleic acid bases at 90% or moreof the 80 nucleotide positions 156, 159, 162, 165, 168, 169, 174, 177,180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217, 219,222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291, 294,297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339,351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402, 405,414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462, 468,483 and 486 of SEQ ID NO:3.
 64. The polynucleotide of claim 61, whereinthe nucleic acid sequence has non-native nucleic acid bases at 95% ormore of the 80 nucleotide positions 156, 159, 162, 165, 168, 169, 174,177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217,219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291,294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336,339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402,405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462,468, 483 and 486 of SEQ ID NO:3.
 65. The polynucleotide of claim 61,wherein the nucleic acid sequence comprises a guanine (g) or a cytosine(c) nucleotide at nucleotide positions 156, 159, 162, 165, 168, 169,174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216,217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285,291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333,336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396,402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459,462, 468, 483 and 486 of SEQ ID NO:3.
 66. The polynucleotide of claim61, wherein the nucleic acid sequence comprises at least 50% GC content.67. The polynucleotide of claim 61, wherein the nucleic acid sequencehas at least 95% sequence identity to nucleotides 145-489 of SEQ ID NO:368. The polynucleotide of claim 61, wherein the nucleic acid sequenceencodes the region of SEQ ID NO:2 that corresponds to mature IL-15. 69.The polynucleotide of claim 68, wherein the nucleic acid sequence has atleast 90% identity to SEQ ID NO:3.
 70. The polynucleotide of claim 61,wherein the polynucleotide comprises a nucleic acid sequence encoding asignal peptide-propeptide (SIG-PRO) or a signal peptide (SIG) from aheterologous protein fused to the nucleic acid sequence encoding theIL-15 protein.
 71. The polynucleotide of claim 70, wherein theheterologous protein is selected from the group consisting ofgranulocyte-macrophage colony stimulating factor (GM-CSF), tissueplasminogen activator (tPA), growth hormone, and an immunoglobulin. 72.The polynucleotide of claim 71, wherein the heterologous protein isGM-CSF.
 73. The polynucleotide of claim 71, wherein the SIG-PRO from theheterologous protein is a tPA SIG PRO having 95% sequence identity toSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:25 or SEQ ID NO:27.
 74. Thepolynucleotide of claim 61, wherein IL-15 production by 293 cellstransfected with the polynucleotide is in an amount at least 5-foldgreater than the amount produced by 293 cells transfected with apolynucleotide comprising the sequence of SEQ ID NO:1, as determined byELISA immunoassay.
 75. An expression vector comprising thepolynucleotide of claim
 61. 76. An isolated host cell comprising thepolynucleotide of claim
 61. 77. An isolated host cell comprising theexpression vector of claim
 75. 78. The isolated host cell of claim 77,wherein the host cell is a mammalian host cell.
 79. The isolated hostcell of claim 78, wherein the mammalian host cell is human.
 80. Theisolated host cell of claim 78, wherein the mammalian host cell is a HEK293-T, COS, CHO, HeLa, NIH3T3, RD or PC12 cell.
 81. The isolated hostcell of claim 79, wherein the mammalian host cell is a HEK293 cell.